TDP-43 causes neurotoxicity and cytoskeletal dysfunction in primary cortical neurons.

Pranetha Baskaran,Sarah Guthrie,Christopher Shaw,Emanuele Buratti

doi:10.1371/journal.pone.0196528

Pranetha Baskaran, Sarah Guthrie + Show 2 more

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https://doi.org/10.1371/journal.pone.0196528

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Journal: PloS one	Publication Date: May 22, 2018
Citations: 28	License type: CC BY 4.0

Affiliation: King's College London

Abstract

TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24–72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy.

Highlights

It is well established that mutations of the TAR DNA-binding protein 43 (TDP-43), a highly conserved nuclear protein, are causal of Amyotrophic Lateral Sclerosis (ALS), and in the most common variant of Frontotemporal Lobe Dementia, FTLD-U [1,2,3,4]
Given that TDP-43 pathology occurs in FTLD-U, which affects the cortex, we determined whether cortical neurons in vitro have the same vulnerability as spinal motor neurons to TDP-43 pathogenicity
In order to evaluate TDP-43 mis-localisation and aggregation, cortical neurons were transfected with a control (EGFP) construct, with a GFP-tagged TDP-43 wild-type (WT) construct, or with M337V or Q331K mutant constructs and fixed at time points from 24 hours to 144 hours

Summary

Introduction

It is well established that mutations of the TAR DNA-binding protein 43 (TDP-43), a highly conserved nuclear protein, are causal of Amyotrophic Lateral Sclerosis (ALS), and in the most common variant of Frontotemporal Lobe Dementia, FTLD-U [1,2,3,4]. Several groups have identified mutations in TDP-43 in sporadic and familial cases of ALS and have provided evidence of a direct link between TDP-43 dysfunction and neurodegeneration[3,4,5]. TDP-43 is predominantly a nuclear protein, it is actively shuttled between the nucleus and the cytoplasm [6] under pathological conditions, TDP-43 is depleted from the nucleus and accumulates in the cytoplasm in both neurons and glia [7, 8].

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